Target - Probable Adenosylhomocysteinase SAHH (S-Adenosyl-L-Homocysteine Hydrolase) (Adohcyase) (Mycobacterium tuberculosis)

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Target (protein/gene name):

PROBABLE ADENOSYLHOMOCYSTEINASE SAHH (S- ADENOSYL-L-HOMOCYSTEINE HYDROLASE) (ADOHCYASE)

NCBI Gene # or RefSeq#:

203282344

Protein ID (NP or XP #) or Wolbachia#:

Rv3248c

Organism (including strain):

Mycobacterium Tuberculosis H37Rv

Etiologic Risk Group (see link below):

3

Background/Disease Information (sort of like the Intro to your Mini Research Write up):

Mycobacterium Tuberculosis is a small, aerobic, bacterial pathogen which causes the disease Tuberculosis. Tuberculosis often affects the lungs and is spread by respiratory fluids which patients transmit through the air. This disease cannot be transmitted be shaking hands, sharing food or drink, touching bed linens or toilet seats, sharing toothbrushes, or kissing.


The symptoms of this disease are chronic cough, bloody sputum, fever, night-sweats, weight loss, no appetite, and chills. This disease is very common throughout the world with more cases occurring in developing countries. Persons with HIV, who have had TB before, drug and alcohol abusers, and those with other health problems are particularly susceptible to contracting Tuberculosis.

S-Asenosyl-L-Homosysteine Hydrolase is a protein present in Mycobacterium Tuberculosis which has functions in cysteine and methionine metabolism and Selenoamino acid metabolism. These are processes which are vital to the survival of the bacterium which, when inhibited, may reduce or stop the effects of the disease Tuberculosis.
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mycobac.jpg

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Link to TDR Targets page (if present):

http://tdrtargets.org/targets/view?gene_id=9870

Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):

https://www.ncbi.nlm.nih.gov/protein/2ZIZ_A

Essentiality of this protein:

Rv3248c has essentiality data
Gene/Ortholog: mtu3307 (OG4_10673); Phenotype: essential; Source study: nmpdr
Gene/Ortholog: sce1759 (OG4_10673); Phenotype: inviable; Source study: yeastgenome
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Larval/Adult Lethal/Arrest; Source study: neb
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Morphology Defect; Source study: neb
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Growth Defect; Source study: neb
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Embryonic Lethal/Arrest; Source study: neb
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Embryonic Lethal/Arrest; Source study: wormbase
Gene/Ortholog: cel11695 (OG4_10673); Phenotype: Larval/Adult Lethal/Arrest; Source study: wormbase
Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford

Complex of proteins:

ternary, with NAD and 3-deazaadenosine

Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):

0.5

*EC#:

3.3.1.1

Link to BRENDA EC# page:

http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.3.1.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Brandaa.jpg




Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):

-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/s_adenosy_l_homocysteine_hydrolase.pdf
-- links to assay reagents (substrates) pages.
Potassium Phosphate
http://www.sigmaaldrich.com/catalog/product/sial/p5379?lang=en&region=US
S-Adenosyl-L-Homocysteine Solution (SAH)
http://www.sigmaaldrich.com/catalog/product/sigma/a9384?lang=en&region=US
Adenosine Deaminase Enzyme Solution (ADA)
http://www.sigmaaldrich.com/catalog/product/sigma/a5168?lang=en&region=US (This is an alternate. Original product was discontinued)
Xanthine Oxidase Enzyme Solution (XO)
http://www.sigmaaldrich.com/catalog/product/sigma/x1875?lang=en&region=US
Nucleoside Phosphorylase Enzyme Solution (NP)
http://www.sigmaaldrich.com/catalog/product/sigma/n3003?lang=en&region=US (Product discontinued)
--- List cost and quantity of substrate reagents, supplier, and catalog #
(see above links)

Structure Available (PDB or Homology model)

-- PDB # or closest PDB entry if using homology model: 2ZIZ

Current Inhibitors:

CHEMBL405186
CHEMBL262063
NORARISTEROMYCIN
CHEMBL261619
CHEMBL129469
CHEMBL49935

Expression Information (has it been expressed in bacterial cells):

Has been expressed in E. Coli
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719076/

Purification Method:

purified as a recombinant His6-tagged protein (HTc-SahH) from Escherichia coli.
(above link)

Image of protein (PyMol with features delineated and shown separately):

2ziz4CHAINS.png2ZIZ4Sticks.png
Figure 1. 2ZIZ protein shown as cartoon colored by chain. Figure 2: 2ZIZ protein shown as sticks and colored by element with carbons as green.

*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):

<span style="background-color: #ffffff; font-family: monospace,serif;">        1 <span class="ff_line">mtgnlvtkns ltpdvrngid fkiadlslad fgrkelriae hempglmslr reyaevqplk</span>
       61 <span class="ff_line">garisgslhm tvqtavliet ltalgaevrw ascnifstqd haaaavvvgp hgtpdepkgv</span>
      121 <span class="ff_line">pvfawkgetl eeywwaaeqm ltwpdpdkpa nmilddggda tmlvlrgmqy ekagvvppae</span>
      181 <span class="ff_line">eddpaewkvf lnllrtrfet dkdkwtkiae svkgvteett tgvlrlyqfa aagdlafpai</span>
      241 <span class="ff_line">nvndsvtksk fdnkygtrhs lidginrgtd aliggkkvli cgygdvgkgc aeamkgqgar</span>
      301 <span class="ff_line">vsvteidpin alqammegfd vvtveeaigd adivvtatgn kdiimlehik amkdhailgn</span>
      361 <span class="ff_line">ighfdneidm aglersgatr vnvkpqvdlw tfgdtgrsii vlsegrllnl gnatghpsfv</span>
      421 <span class="ff_line">msnsfanqti aqielwtknd eydnevyrlp khldekvari hvealgghlt kltkeqaeyl</span>
      481 <span class="ff_line">gvdvegpykp dhyry</span></span>

Length of your protein in Amino Acids: 495

Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:

<span style="background-color: #ffffff; color: #626262;">54323.6</span>

Molar Extinction coefficient of your protein at 280 nm wavelength:

<span style="background-color: #ffffff; color: #626262;">Ext. coefficient    67505
Abs 0.1% (=1 g/l)   1.243, assuming all pairs of Cys residues form cystines
 
 
Ext. coefficient    67380
Abs 0.1% (=1 g/l)   1.240, assuming all Cys residues are reduced
</span>

TMpred graph Image:

results
results

*CDS Gene Sequence (paste as text only):

<span style="background-color: #ffffff; font-family: monospace,serif;"><span class="ff_line">TCAGTAGCGGTAGTGGTCCGGCTTGTAGGGACCCTCGACGTCGACGCCGAGGTATTCGGCCTGCTCCTTG
GTCAGCTTGGTCAGGTGACCGCCAAGGGCCTCGACATGGATTCGAGCCACCTTCTCGTCGAGGTGCTTGG
GCAGCCGGTACACCTCGTTGTCGTACTCGTCGTTCTTGGTCCACAGCTCGATCTGGGCGATCGTCTGGTT
AGCGAAGCTGTTGCTCATCACGAACGAGGGGTGCCCGGTGGCATTGCCCAGGTTCAGCAGCCGCCCCTCG
GACAGCACGATGATCGAGCGGCCCGTGTCGCCAAAGGTCCACAGGTCGACCTGAGGCTTGACGTTGACCC
GTGTCGCCCCGGAGCGCTCCAGCCCGGCCATGTCGATCTCGTTGTCGAAGTGGCCGATATTTCCCAGGAT
CGCGTGGTCCTTCATCGCCTTAATGTGCTCGAGCATGATGATGTCTTTGTTGCCGGTCGCGGTTACGACG
ATGTCGGCGTCCCCGATGGCCTCCTCGACGGTGACCACGTCGAAGCCCTCCATCATGGCCTGCAGCGCGT
TGATCGGGTCGATCTCGGTGACGGAGACCCGCGCTCCCTGGCCCTTCATCGCCTCCGCACAGCCCTTACC
GACGTCGCCGTAGCCGCAGATGAGGACCTTCTTACCGCCGATCAGCGCGTCGGTGCCGCGGTTGATGCCG
TCGATCAGGGAGTGCCGAGTGCCGTACTTGTTGTCGAATTTGGACTTGGTCACCGAGTCGTTGACGTTGA
TCGCCGGGAAGGCCAGATCCCCGGCCGCGGCGAATTGGTAGAGCCGCAGCACGCCGGTGGTGGTCTCCTC
GGTGACGCCCTTGACCGACTCGGCTATCTTGGTCCACTTGTCCTTGTCGGTCTCGAAGCGGGTCCGTAGC
AGGTTCAGGAAGACCTTCCACTCGGCGGGGTCGTCCTCCTCGGCGGGCGGCACCACGCCGGCCTTCTCAT
ACTGCATGCCGCGCAGCACCAACATGGTGGCGTCACCGCCGTCATCGAGGATCATGTTGGCCGGCTTGTC
GGGGTCCGGCCAGGTGAGCATCTGCTCGGCGGCCCACCAGTACTCTTCGAGCGTCTCGCCCTTCCACGCG
AACACCGGGACACCCTTGGGCTCGTCGGGGGTGCCGTGCGGGCCGACCACGACGGCGGCGGCGGCGTGAT
CCTGGGTGGAGAAGATGTTGCACGAGGCCCAGCGGACTTCGGCGCCCAGCGCGGTGAGGGTTTCGATCAA
CACCGCGGTCTGCACCGTCATGTGCAGCGAACCCGAGATCCGGGCCCCCTTCAGGGGTTGCACCTCGGCA
TACTCGCGCCGCAGCGACATCAGGCCGGGCATCTCGTGCTCGGCGATCCGGAGTTCTTTGCGGCCGAAAT
CCGCTAGTGACAGGTCGGCGATCTTAAAGTCGATGCCGTTACGAACGTCAGGGGTCAGCGAATTTTTGGT
CACCAAATTTCCGGTCAT</span></span>

GC% Content for gene:

Not Available

CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):

Not Available

GC% Content for gene (codon optimized):

Not Available

Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.

Primer design results for 'tail' primers (this is just 2 sequences)